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Supplementary Figure S2. Correlation between plasma RTK changes with MAPKi, PFS, and initial tumor shrinkage (using RECIST) in melanoma patients.

Supplementary Methods, Figure Legends

Supplementary Figures 1-2 from Three-kinase inhibitor combination recreates multipathway effects of a geldanamycin analogue on hepatocellular carcinoma cell death

Multitarget compounds that act on a diverse set of regulatory pathways are emerging as a therapeutic approach for a variety of cancers. Toward a more specified use of this approach, we hypothesize that the desired efficacy can be recreated in terms of a particular combination of relatively more specific (i.e., ostensibly single target) compounds. We test this hypothesis for the geldanamycin analogue 17-Allylamino-17-demethoxygeldanamycin (17AAG) in hepatocellular carcinoma cells, measuring critical phosphorylation levels that indicate the kinase pathway effects correlating with apoptotic responsiveness of the Hep3B cell line in contrast to the apoptotic resistance of the Huh7 cell line. A principal components analysis (PCA) constructed from time course measurements of seven phosphoprotein signaling levels identified modulation of the AKT, IκB kinase, and signal transducer and activator of transcription 3 pathways by 17AAG treatment as most important for distinguishing these cell-specific death responses. The analysis correctly suggested from 17AAG-induced effects on these phosphoprotein levels that the FOCUS cell line would show apoptotic responsiveness similarly to Hep3B. The PCA also guided the inhibition of three critical pathways and rendered Huh7 cells responsive to 17AAG. Strikingly, in all three hepatocellular carcinoma lines, the three-inhibitor combination alone exhibited similar or greater efficacy to 17AAG. We conclude that (a) the PCA captures and clusters the multipathway phosphoprotein time courses with respect to their 17AAG-induced apoptotic responsiveness and (b) we can recreate, in a more specified manner, the cellular responses of a prospective multitarget cancer therapeutic. [Mol Cancer Ther 2009;8(8):2183–92]

In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influence phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHH) cultured in a commercially available perfused bioreactor. A 3D printing-based alginate microwell system was designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver were engineered to replace polystyrene scaffolds, and the biochemical microenvironment was modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increased tissue density, albumin secretion, and CYP3A4 activity but also upregulated inflammatory markers. Basal inflammatory markers were lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibited the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA-seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro.

On-platform pumping systems are a potentially critical technology for microphysiological systems (MPS) to control the pressure and flow of growth media. Supporting sufficient physiological tissue quantity in culture requires fluid flow rates on the order of microliters per second, which is larger than for typical microfluidic systems. Thus, a need exists for new types of pumping systems operating in this flow range while maintaining stringent sterility of the culture as well as enabling tight temperature control at ${37}^{\circ } {\rm C}$ . Flow rates and pressure also need to be readily computer-controlled, with a manageable set of connections into the culture incubator environment. This article describes a novel mesoscale electromagnetically driven pumping system designed to meet all these requirements. Our design achieves a very low energy dissipation of ${0.65}\,{\rm mJ}$ per switching event, which allows one pumping channel to operate at ${0.45}\,{{\rm {\mu } L} /{\rm s}}$ with an average power dissipation of 1.3 mW and ${0.04}\,{{^\circ } {\rm C}}$ temperature rise in each actuator. The actuator operates in a bistable, teeter-totter configuration with a latching force of ${4.5}\,{\rm N}$ , and a relatively large stroke of ${400}\,{{\mu } {\rm m}}$ at the actuator pole face. Preliminary operational pumping test results show the potential of this type of electromagnetic actuator for fluidic pumping. Due to their compact configuration and very high energy efficiency, these pumps can provide the foundation for multichannel, on-platform pumping for MPS platforms, as well as for a range of sterile, temperature-sensitive microflow devices such as portable, battery-operated insulin pumps.

Background Pediatric gliomas comprise a diverse set of brain tumor entities that have substantial long-term ramifications for patient survival and quality of life. However, the study of these tumors is currently limited due to a lack of authentic models. Additionally, many aspects of pediatric brain tumor biology, such as tumor cell invasiveness, have been difficult to study with currently available tools. To address these issues, we developed a synthetic extracellular matrix (sECM)-based culture system to grow and study primary pediatric brain tumor cells. Methods We developed a brain-like sECM material as a supportive scaffold for the culture of primary, patient-derived pediatric glioma cells and established patient-derived cell lines. Primary juvenile brainstem-derived murine astrocytes were used as a feeder layer to support the growth of primary human tumor cells. Results We found that our culture system facilitated the proliferation of various primary pediatric brain tumors, including low-grade gliomas, and enabled ex vivo testing of investigational therapeutics. Additionally, we found that tuning this sECM material allowed us to assess high-grade pediatric glioma cell invasion and evaluate therapeutic interventions targeting invasive behavior. Conclusion Our sECM culture platform provides a multipurpose tool for pediatric brain tumor researchers that enables both a wide breadth of biological assays and the cultivation of diverse tumor types.

An estimated 70% of individuals with type 2 diabetes also suffer from nonalcoholic fatty liver disease (NAFLD) . Insulin resistance is pathologically fundamental to both NAFLD and T2D, however the mechanisms of insulin resistance linking these complex diseases remains incompletely understood. Microphysiological systems provide an avenue to simulate disease parameters in the context of human biology, however current in vitro models of hepatic insulin resistance often include nutrient and insulin concentrations orders of magnitude above in vivo levels. Here, we established an in vitro 3D model culturing primary human hepatocytes and non-parenchymal cells in physiologically relevant media conditions simulating both baseline physiological and T2D conditions. Albumin secretion and cytochrome p450 assays indicate hepatocytes remain functional over two weeks in culture for both physiological and T2D conditions. Results further show hepatocytes cultured in physiological conditions maintain insulin responsiveness after 12 days in culture, measured by glucose output following dose response of insulin stimulation. Conversely, cells cultured in T2D media conditions have significantly attenuated response to insulin stimulation compared to the physiological condition. Measuring insulin clearance from the media also showed reduced clearance over time for the T2D conditions. Together, these results demonstrate the induction of hepatic insulin resistance through culture with insulin concentrations found in the portal vein of T2D patients. Studies further applying this in vitro model to understand insulin resistance mechanisms are ongoing. Disclosure E.Tevonian: Research Support; Novo Nordisk. J.F.Jeppesen: Employee; Novo Nordisk A/S. D.Lauffenburger: None. L.G.Griffith: Research Support; Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S.

Intestinal homeostasis is tightly regulated by the orchestrated actions of a multitude of cell types, including enterocytes, goblet cells and immune cells. Disruption of intestinal barrier function can increase susceptibility to pathogen invasion and destabilize commensal microbial-epithelial-immune interaction, manifesting in various intestinal and systemic pathologies. However, a quantitative understanding of how these cell types communicate and collectively contribute to tissue function in health and disease is lacking. Here, we utilized a human intestinal epithelial-dendritic cell model and multivariate analysis of secreted factors to investigate the cellular crosstalk in response to physiological and/or pathological cues (e.g., endotoxin, non-steroidal anti-inflammation drug (NSAID)). Specifically, we demonstrated that treatment with diclofenac (DCF), an NSAID commonly used to treat inflammation associated with acute infection and other conditions, globally suppressed cytokine secretion when dosed in isolation. However, the disruption of barrier function induced by DCF allowed for luminal lipopolysaccharide (LPS) translocation and activation of resident immune cells that overrode the anti-inflammatory influence of DCF. DCF-facilitated inflammation in the presence of LPS was in part mediated by upregulation of macrophage migration inhibitory factor (MIF), an important regulator of innate immunity. However, while neutralization of MIF activity normalized inflammation, it did not lead to intestinal healing. Our data suggest that systems-wide suppression of inflammation alone is insufficient to achieve mucosal healing, especially in the presence of DCF, the target of which, the COX-prostaglandin pathway, is central to mucosal homeostasis. Indeed, DCF removal post-injury enabled partial recovery of intestinal epithelium functions, and this recovery phase was associated with upregulation of a subset of cytokines and chemokines, implicating their potential contribution to intestinal healing. The results highlight the utility of an intestinal model capturing immune function, coupled with multivariate analysis, in understanding molecular mechanisms governing response to microbial factors, supporting application in studying host-pathogen interactions.

STUDY OBJECTIVE To assess the feasibility of a noncontact radio sensor as an objective measurement tool to study postoperative recovery from endometriosis surgery. DESIGN Prospective cohort pilot study. SETTING Center for minimally invasive gynecologic surgery at an academically affiliated community hospital in conjunction with in-home monitoring. PATIENTS Patients aged above 18 years who sleep independently and were scheduled to have laparoscopy for the diagnosis and treatment of suspected endometriosis. INTERVENTIONS A wireless, noncontact sensor, Emerald, was installed in the subjects' home and used to capture physiologic signals without body contact. The device captured objective data about the patients' movement and sleep in their home for 5 weeks before surgery and approximately 5 weeks postoperatively. The subjects were concurrently asked to complete a daily pain assessment using a numeric rating scale and a free text survey about their daily symptoms. MEASUREMENTS AND MAIN RESULTS Three women aged 23 years to 39 years and with mild to moderate endometriosis participated in the study. Emerald-derived sleep and wake times were contextualized and corroborated by select participant comments from retrospective surveys. In addition, self-reported pain levels and 1 sleep variable, sleep onset to deep sleep time, showed a significant (p

SummaryThe capacity to generate functional hepatocytes from renewable human pluripotent stem cells (hPSCs) could address limited supplies of primary human hepatocytes. However, hepatocytes differentiated from hPSCs in vitro are functionally immature. To understand mechanisms regulating maturation of in vitro derived hepatocytes, we developed a 3D spheroid differentiation system and compared gene regulatory elements in uncultured human primary hepatocytes with those in hepatocytes that were differentiated in 2D or 3D conditions from human PSCs by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Three-dimensional differentiation improved enhancer activity and expression of transcription factor ONECUT1, but was insufficient to upregulate human-specific mature hepatocytes marker gene CYP3A4 or super-enhancer regulated transcription factor gene NFIC. Regulome comparisons showed reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and in active enhancers without reduced transcription of THRB, suggesting the regulation at the level of THRB ligands in PSC-differentiated hepatocytes. Addition of thyroid hormone T3 to the PSC-differentiated hepatocytes increased CYP3A4 expression. T3 increased binding of THRB to the CYP3A4 proximal enhancer and restored the super-enhancer status and gene expression of NFIC and reduced expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the 3D PSC-hepatocytes into immunocompromised mice resulted in engraftment of human hepatocytes in the mouse liver parenchyma without disrupting normal liver histology at 6 months after transplantation. This work provides insights into the functions of nuclear receptor THRB and highlights the importance of the environmental factors-nuclear receptors axis in regulating maturation of human PSC-differentiated cell types.

© 2020 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Adenomyosis remains an enigmatic disease in the clinical and research communities. The high prevalence, diversity of morphological and symptomatic presentations, array of potential etiological explanations, and variable response to existing interventions suggest that different subgroups of patients with distinguishable mechanistic drivers of disease may exist. These factors, combined with the weak links to genetic predisposition, make the entire spectrum of the human condition challenging to model in animals. Here, after an overview of current approaches, a vision for applying physiomimetic modeling to adenomyosis is presented. Physiomimetics combines a system's biology analysis of patient populations to generate hypotheses about mechanistic bases for stratification with in vitro patient avatars to test these hypotheses. A substantial foundation for three-dimensional (3D) tissue engineering of adenomyosis lesions exists in several disparate areas: epithelial organoid technology; synthetic biomaterials matrices for epithelial-stromal coculture; smooth muscle 3D tissue engineering; and microvascular tissue engineering. These approaches can potentially be combined with microfluidic platform technologies to model the lesion microenvironment and can potentially be coupled to other microorgan systems to examine systemic effects. In vitro patient-derived models are constructed to answer specific questions leading to target identification and validation in a manner that informs preclinical research and ultimately clinical trial design.

Research question What is the association between endometrioma-affected ovaries, their follicular fluid inflammatory microenvironment, and ovary-specific oocyte and embryo yield and quality? Design Exposure-matched prospective cohort study conducted at a university-affiliated infertility clinic. Thirty-four women presenting for oocyte retrieval were enrolled between 2012 and 2013: women with unilateral endometrioma and no other observed peritoneal or deep lesions (n = 10) and women with no signs or symptoms of endometriosis (n = 24). Follicular fluid was aspirated at the time of oocyte retrieval. Samples from each ovary were analysed using a 27-plex immunoassay panel. The associations were evaluated by ovary-specific endometrioma exposure status (affected, unaffected, unexposed) with cytokine levels, oocyte yield and embryo quality. Results Levels of interleukin (IL)-8 and monocyte chemoattractant protein-1 were higher in fluid obtained from endometrioma-affected ovaries compared with the unexposed ovaries from women without endometriosis, with intermediate levels observed in the contralateral unaffected ovaries. More modest differences were observed for IL-1β and IL-6. The affected ovaries of women with endometriosis yielded fewer oocytes (mean ± SD = 4.6 ± 2.3) compared with both the unaffected (6.0 ± 3.8) and unexposed (7.9 ± 5.6) ovaries. After adjusting for potential confounders and variables generated in a cytokine principal components analysis, oocyte yield remained slightly lower for the endometrioma-affected ovaries compared with unexposed ovaries. No informative differences among ovary groups for embryo quality parameters were observed. Conclusions The results suggest that the inflammatory milieu of ovarian endometriosis is strongly localized and has a more modestly systemic effect. The effect of endometriomas on infertility, however, cannot be entirely explained by increased inflammation.

Endometriosis remains a challenge to understand and to diagnose. This is an observational cross-sectional pilot study to characterize the gut and vaginal microbiome profiles among endometriosis patients and control subjects without the disease and to explore their potential use as a less-invasive diagnostic tool for endometriosis. Overall, 59 women were included, n = 35 with endometriosis and n = 24 controls. Rectal and vaginal samples were collected in two different periods of the menstrual cycle from all subjects. Gut and vaginal microbiomes from patients with different rASRM (revised American Society for Reproductive Medicine) endometriosis stages and controls were analyzed. Illumina sequencing libraries were constructed using a two-step 16S rRNA gene PCR amplicon approach. Correlations of 16S rRNA gene amplicon data with clinical metadata were conducted using a random forest-based machine-learning classification analysis. Distribution of vaginal CSTs (community state types) significantly differed between follicular and menstrual phases of the menstrual cycle (p = 0.021, Fisher’s exact test). Vaginal and rectal microbiome profiles and their association to severity of endometriosis (according to rASRM stages) were evaluated. Classification models built with machine-learning methods on the microbiota composition during follicular and menstrual phases of the cycle were built, and it was possible to accurately predict rASRM stages 1–2 verses rASRM stages 3–4 endometriosis. The feature contributing the most to this prediction was an OTU (operational taxonomic unit) from the genus Anaerococcus. Gut and vaginal microbiomes of women with endometriosis have been investigated. Our findings suggest for the first time that vaginal microbiome may predict stage of disease when endometriosis is present. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s43032-019-00113-5) contains supplementary material, which is available to authorized users.

The human endometrium is a mucosal barrier that undergoes cycles of growth, differentiation, and breakdown in response to sex hormone fluctuations. Dynamic tissue responses to hormones are primarily driven by epithelial-stromal communication and its dysregulation is linked to myriad gynecological disorders. The lack of robust in vitro models for the long-term 3D co-culture of patient-derived endometrial epithelial and stromal cells hinders dissection of this crosstalk and thus impairs progress in disease treatment. Here, we describe a versatile synthetic extracellular matrix tailored to the endometrium that enables the in vitro modeling of human healthy and disease states across the menstrual cycle. We used a tissue-inspired approach to semi-empirically screen a parameter space that encompasses the biophysical and molecular features of the endometrial microenvironment. Leveraging cell-specific integrin expression profiles, we defined a modular polyethylene glycol (PEG)-based hydrogel that fosters hormone-driven expansion and differentiation of epithelial organoids co-cultured with stromal cells. Characteristic morphological and molecular responses of each cell type to hormone changes were observed when cells were co-encapsulated in hydrogels tuned to a stiffness regime similar to the native tissue and functionalized with a collagen-derived adhesion peptide (GFOGER) and a fibronectin-derived peptide (PHSRN-K-RGD). Using transcriptomic and functional assays, we demonstrate the systems ability to recapitulate menstrual-cycle specific reproductive events and identified that inflammation-induced dysregulation of epithelial proliferation is mediated via the stromal compartment. Altogether, we demonstrate the development of a fully synthetic matrix to sustain the dynamic changes of the endometrial microenvironment and support its applications to understand endometriotic diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/462577v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@132d3fborg.highwire.dtl.DTLVardef@1fb2841org.highwire.dtl.DTLVardef@ff18dorg.highwire.dtl.DTLVardef@bb9d8f_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Organoid cultures are proving to be powerful in vitro models that closely mimic the cellular constituents of their native tissue. Organoids are typically expanded and cultured in a 3D environment using either naturally derived or synthetic extracellular matrices. Assessing the morphology and growth characteristics of these cultures has been difficult due to the many imaging artifacts that accompany the corresponding images. Unlike single cell cultures, there are no reliable automated segmentation techniques that allow for the localization and quantification of organoids in their 3D culture environment. Here we describe OrgaQuant, a deep convolutional neural network implementation that can locate and quantify the size distribution of human intestinal organoids in brightfield images. OrgaQuant is an end-to-end trained neural network that requires no parameter tweaking; thus, it can be fully automated to analyze thousands of images with no user intervention. To develop OrgaQuant, we created a unique dataset of manually annotated human intestinal organoid images with bounding boxes and trained an object detection pipeline using TensorFlow. We have made the dataset, trained model and inference scripts publicly available along with detailed usage instructions.

Experimental in vitro models that accurately capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix, specifically designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from murine genetically engineered models and human patients, essential adhesive cues were empirically defined and incorporated into the hydrogel scaffold, revealing a functional role of laminin – integrin a3/a6 signalling in adhesion and survival of pancreatic organoids. Altered tissue stiffness — a hallmark of pancreatic cancer — was recapitulated in culture by adjusting the hydrogel properties to engage mechano-signalling and alter organoid growth. Pancreatic stromal cells were readily incorporated and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro., This upload is compiled of 21 individual data packages which contain the raw data and R-scripts necessary to generate the outputs displayed in the paper. Each folder contains a README document which assists the reader through the files.

© 2020 The Authors Women's health concerns are generally underrepresented in basic and translational research, but reproductive health in particular has been hampered by a lack of understanding of basic uterine and menstrual physiology. Menstrual health is an integral part of overall health because between menarche and menopause, most women menstruate. Yet for tens of millions of women around the world, menstruation regularly and often catastrophically disrupts their physical, mental, and social well-being. Enhancing our understanding of the underlying phenomena involved in menstruation, abnormal uterine bleeding, and other menstruation-related disorders will move us closer to the goal of personalized care. Furthermore, a deeper mechanistic understanding of menstruation—a fast, scarless healing process in healthy individuals—will likely yield insights into a myriad of other diseases involving regulation of vascular function locally and systemically. We also recognize that many women now delay pregnancy and that there is an increasing desire for fertility and uterine preservation. In September 2018, the Gynecologic Health and Disease Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a 2-day meeting, “Menstruation: Science and Society” with an aim to “identify gaps and opportunities in menstruation science and to raise awareness of the need for more research in this field.” Experts in fields ranging from the evolutionary role of menstruation to basic endometrial biology (including omic analysis of the endometrium, stem cells and tissue engineering of the endometrium, endometrial microbiome, and abnormal uterine bleeding and fibroids) and translational medicine (imaging and sampling modalities, patient-focused analysis of menstrual disorders including abnormal uterine bleeding, smart technologies or applications and mobile health platforms) to societal challenges in health literacy and dissemination frameworks across different economic and cultural landscapes shared current state-of-the-art and future vision, incorporating the patient voice at the launch of the meeting. Here, we provide an enhanced meeting report with extensive up-to-date (as of submission) context, capturing the spectrum from how the basic processes of menstruation commence in response to progesterone withdrawal, through the role of tissue-resident and circulating stem and progenitor cells in monthly regeneration—and current gaps in knowledge on how dysregulation leads to abnormal uterine bleeding and other menstruation-related disorders such as adenomyosis, endometriosis, and fibroids—to the clinical challenges in diagnostics, treatment, and patient and societal education. We conclude with an overview of how the global agenda concerning menstruation, and specifically menstrual health and hygiene, are gaining momentum, ranging from increasing investment in addressing menstruation-related barriers facing girls in schools in low- to middle-income countries to the more recent “menstrual equity” and “period poverty” movements spreading across high-income countries.

Summary Background The gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a long-term co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. Methods Here, we investigate the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a gut microbiome (GuMI) physiome platform that we designed and fabricated. Findings Long-term continuous co-culture of F. prausnitzii for 2 days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, results in a strictly anaerobic apical environment fostering the growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identify elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrate the anti-inflammatory effects of F. prausnitzii through histone deacetylase (HDAC) and the Toll-like receptor-TLR-nuclear factor κB (NF-κB) axis. Finally, we identify that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. Conclusions Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies using this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria. Funding US National Institute of Biomedical Imaging and Bioengineering, Boehringer Ingelheim Strategic Hub for Innovative New Therapeutics Concept Exploration (SHINE) Program, US National Institute of Environmental Health Sciences.

PROBLEM To compare inflammatory- and immune-associated peritoneal cytokines of adolescents and adults with and without endometriosis. METHODS OF STUDY In a nested case-control study in multiple university-affiliated scientific centers, ten adolescents and thirteen adults with visually and histologically confirmed endometriosis (cases), thirteen adolescents with visually suspected endometriosis but indeterminate (seven patients) or negative (six patients) histology, and fifteen adults undergoing surgery for non-malignant gynecologic disease without endometriosis (controls) underwent laparoscopic aspiration of peritoneal fluid (PF), from which PF and conditioned medium (CM) cytokine levels were assayed. RESULTS Compared to adults with endometriosis, MCP-3, IL-12p40, MIP-1β, and IL-15 were significantly higher among adolescents with endometriosis, while TNF-β and CTACK were lower among adolescents. These differences were similar comparing adolescents with endometriosis to adult controls except for MIP-1β, which was not statistically different. MIP-1β was, however, the only cytokine observed to differ between adult cases and controls. There were no significant differences in CM cytokines among the three groups. Results were similar when analyses were restricted to samples collected (a) during menstrual cycle days 1-10, (b) from patients unexposed to exogenous hormones, or (c) from all adolescents despite presence or absence of histologic endometriosis. CONCLUSION Biologically relevant and statistically significant differences in six PF cytokines were observed and suggest a more pro-invasion cytokine profile among adolescents with endometriosis. Adolescents with endometriosis have unique peritoneal cytokine profiles and molecular behavior when compared to adults with and without endometriosis.

Engineered model of the human gut-liver-brain axis can be used to study the link between the microbiome and Parkinson’s disease., Slow progress in the fight against neurodegenerative diseases (NDs) motivates an urgent need for highly controlled in vitro systems to investigate organ-organ– and organ-immune–specific interactions relevant for disease pathophysiology. Of particular interest is the gut/microbiome-liver-brain axis for parsing out how genetic and environmental factors contribute to NDs. We have developed a mesofluidic platform technology to study gut-liver-cerebral interactions in the context of Parkinson’s disease (PD). It connects microphysiological systems (MPSs) of the primary human gut and liver with a human induced pluripotent stem cell–derived cerebral MPS in a systemically circulated common culture medium containing CD4+ regulatory T and T helper 17 cells. We demonstrate this approach using a patient-derived cerebral MPS carrying the PD-causing A53T mutation, gaining two important findings: (i) that systemic interaction enhances features of in vivo–like behavior of cerebral MPSs, and (ii) that microbiome-associated short-chain fatty acids increase expression of pathology-associated pathways in PD.

SummaryAssociation between the microbiome, IBD and liver diseases are known, yet cause and effect remain elusive. By connecting human microphysiological systems of the gut, liver and circulating Treg/Th17 cells, we modeled progression of ulcerative colitis (UC) ex vivo. We show that microbiome-derived short-chain fatty acids (SCFA) may either improve or worsen disease severity, depending on the activation state of CD4 T cells. Employing multiomics, we found SCFA increased production of ketone bodies, glycolysis and lipogenesis, while markedly reducing innate immune activation of the UC gut. However, during acute T cell-mediated inflammation, SCFA exacerbated CD4+T cell effector function, partially through metabolic reprograming, leading to gut barrier disruption and hepatic injury. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity, metabolism and tissue homeostasis.

A clinically relevant risk factor forClostridioides difficile-associated dis-ease (CDAD) is recent antibiotic treatment. Although broad-spectrum antibioticshave been shown to disrupt the structure of the gut microbiota, some antibioticsappear to increase CDAD risk without being highly active against intestinal anaerobes, suggesting direct nonantimicrobial effects. We examined cell biological effectsof antibiotic exposure that may be involved in bacterial pathogenesis using aninvitrogermfree human colon epithelial culture model. We found a marked loss of mucosal barrier and immune function with exposure to the CDAD-associated antibiotics clindamycin and ciprofloxacin, distinct from the results of pretreatment with anantibiotic unassociated with CDAD, tigecycline, which did not reduce innate immuneor mucosal barrier functions. Importantly, pretreatment with CDAD-associated antibi-otics sensitized mucosal barriers to C. difficiletoxin activity in primary cell-derived enteroid monolayers. These data implicate commensal-independent gut mucosal barrier changes in the increased risk of CDAD with specific antibiotics and warrantfurther studies inin vivosystems. We anticipate this work to suggest potential ave-nues of research for host-directed treatment and preventive therapies for CDAD., National Institutes of Health (U.S.) (Grant 01-EB021908), National Cancer Institute (U.S.) (Grant P30-ES002109), David H. Koch Institute for Integrative Cancer Research at MIT (Support core Grant P30-CA14501)

Epithelial organoids derived from human donor tissues are important tools in fields ranging from regenerative medicine to drug discovery. Organoid culture requires expansion of stem/progenitor cells in Matrigel, a tumor-derived extracellular matrix (ECM). An alternative completely synthetic ECM could improve reproducibility, clarify mechanistic phenomena, and enable human implantation of organoids. We designed synthetic ECMs with tunable biomolecular and biophysical properties to identify gel compositions supporting human tissue-derived stem/progenitor epithelial cells as enteroids and organoids starting with single cells rather than tissue fragments. The synthetic ECMs consist of 8-arm PEG-macromers modified with ECM-binding peptides and different combinations of integrin-binding peptides, crosslinked with peptides susceptible to matrix metalloprotease (MMP) degradation, and tuned to exhibit a range of biophysical properties. A gel containing an α2β1 integrin-binding peptide (GFOGER) and matrix binder peptides grafted to a 20 kDa 8-arm PEG macromer showed the most robust support of human duodenal and colon enteroids and endometrial organoids. In this synthetic ECM, human intestinal enteroids and endometrial organoids emerge from single cells and show cell-specific and apicobasal polarity markers upon differentiation. Intestinal enteroids, in addition, retain their proliferative capacity, are functionally responsive to basolateral stimulation, express canonical markers of intestinal crypt cells including Paneth cells, and can be serially passaged. The success of this synthetic ECM in supporting human postnatal organoid culture from multiple different donors and from both the intestine and endometrium suggests it may be broadly useful for other epithelial organoid culture.

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

Expanding the toolkit of modular and functional synthetic material systems for biomimetic extracellular matrices (ECMs) is needed for achieving more predictable and characterizable cell culture. In the present study, we engineered a synthetic hydrogel system incorporating poly(γ-propargyl-l-glutamate) (PPLG), an N-carboxy anhydride polypeptide with a unique α-helical secondary structure. PPLG macromers were cross-linked into poly(ethylene glycol) (PEG) networks to form hybrid polypeptide-PEG hydrogels. We compared the properties of PPLG-PEG to systems where the PPLG macromers were replaced with 8-arm PEG or poly(γ-propargyl-d,l-glutamate) (PPDLG), which has a flexible random-coil conformation. We evaluated each hydrogel system as synthetic ECMs for two-dimensional (2D) endothelial cell culture. Cells on PPLG-PEG displayed superior attachment and spreading at comparable adhesion ligand incorporation concentrations, demonstrating the unique benefit of combining the more rigid and hydrophobic α-helical PPLG within the more flexible and hydrophilic PEG matrix. The modular PPLG macromer is a promising building block for developing other types of PPLG-based hydrogels with favorable and tunable properties.

© 2017 The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. Keywords: Microphysiologic systems; Organoids; 3D culture systems, National Institutes of Health (U.S.) (Grant UH3TR000496), National Institutes of Health (U.S.) (Grant UH3TR000503)

Background: Increases in expression of ADAM10 and ADAM17 genes and proteins are inconsistently found in cancer lesions, and are not validated as clinically useful biomarkers. The enzyme-specific proteolytic activities, which are solely mediated by the active mature enzymes, directly reflect enzyme cellular functions and might be superior biomarkers than the enzyme gene or protein expressions, which comprise the inactive proenzymes and active and inactivated mature enzymes. Methods: Using a recent modification of the proteolytic activity matrix analysis (PrAMA) measuring specific enzyme activities in cell and tissue lysates, we examined the specific sheddase activities of ADAM10 (ADAM10sa) and ADAM17 (ADAM17sa) in human non-small cell lung-carcinoma (NSCLC) cell lines, patient primary tumors and blood exosomes, and the noncancerous counterparts. Results: NSCLC cell lines and patient tumors and exosomes consistently showed significant increases of ADAM10sa relative to their normal, inflammatory and/or benign-tumor controls. Additionally, stage IA-IIB NSCLC primary tumors of patients who died of the disease exhibited greater increases of ADAM10sa than those of patients who survived 5 years following diagnosis and surgery. In contrast, NSCLC cell lines and patient tumors and exosomes did not display increases of ADAM17sa. Conclusions: This study is the first to investigate enzyme-specific proteolytic activities as potential cancer biomarkers. It provides a proof-of-concept that ADAM10sa could be a biomarker for NSCLC early detection and outcome prediction. To ascertain that ADAM10sa is a useful cancer biomarker, further robust clinical validation studies are needed., National Institutes of Health (U.S.) (R01 CA96504)

In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influences phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHHs) cultured in a commercially available perfused bioreactor. A 3D printing‐based alginate microwell system is designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver are engineered to replace polystyrene scaffolds, and the biochemical microenvironment is modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increases tissue density, albumin secretion, and CYP3A4 activity but also upregulates inflammatory markers. Basal inflammatory markers are lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibit the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA‐Seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro.

Microphysiological systems (MPS), comprising human cell cultured in formats that capture features of the three-dimensional (3D) microenvironments of native human organs under microperfusion, are promising tools for biomedical research. Here we report the development of a mesoscale physiological system (MePS) enabling the long-term 3D perfused culture of primary human hepatocytes at scales of over 106 cells per MPS. A central feature of the MePS, which employs a commercially-available multiwell bioreactor for perfusion, is a novel scaffold comprising a dense network of nano- and micro-porous polymer channels, designed to provide appropriate convective and diffusive mass transfer of oxygen and other nutrients while maintaining physiological values of shear stress. The scaffold design is realized by a high resolution stereolithography fabrication process employing a novel resin. This new culture system sustains mesoscopic hepatic tissue-like cultures with greater hepatic functionality (assessed by albumin and urea synthesis, and CYP3A4 activity) and lower inflammation markers compared to comparable cultures on the commercial polystyrene scaffold. To illustrate applications to disease modeling, we established an insulin-resistant phenotype by exposing liver cells to hyperglycemic and hyperinsulinemic media. Future applications of the MePS include the co-culture of hepatocytes with resident immune cells and the integration with multiple organs to model complex liver-associated diseases

Methods to parse paracrine epithelial-stromal communication networks are a vital need in drug development, as disruption of these networks underlies diseases ranging from cancer to endometriosis. Here, we describe a modular, synthetic, and dissolvable extracellular matrix (MSD-ECM) hydrogel that fosters functional 3D epithelial-stromal co-culture, and that can be dissolved on-demand to recover cells and paracrine signaling proteins intact for subsequent analysis. Specifically, synthetic polymer hydrogels, modified with cell-interacting adhesion motifs and crosslinked with peptides that include a substrate for cell-mediated proteolytic remodeling, can be rapidly dissolved by an engineered version of the microbial transpeptidase Sortase A (SrtA) if the crosslinking peptide includes a SrtA substrate motif and a soluble second substrate. SrtA-mediated dissolution affected only 1 of 31 cytokines and growth factors assayed, whereas standard protease degradation methods destroyed about half of these same molecules. Using co-encapsulated endometrial epithelial and stromal cells as one model system, we show that the dynamic cytokine and growth factor response of co-cultures to an inflammatory cue is richer and more nuanced when measured from SrtA-dissolved gel microenvironments than from the culture supernate. This system employs accessible, reproducible reagents and facile protocols; hence, has potential as a tool in identifying and validating therapeutic targets in complex diseases., National Institutes of Health (U.S.) (R01EB010246), National Institutes of Health (U.S.) (UH2TR000496), Institute for Collaborative Biotechnologies (W911NF-09-0001), National Institutes of Health (U.S.) (T32GM008334), United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), John and Karinne Begg Fund, Begg New Horizon Fund for Undergraduate Research at MIT, Massachusetts Institute of Technology. Biophysical Instrumentation Facility, Manton Foundation, Ludwig Postdoctoral Fellowship for Cancer Research, Swiss National Science Foundation (Postdoctoral Fellowship)

Microphysiological systems (MPS) provide relevant physiological environments in vitro for studies of pharmacokinetics, pharmacodynamics and biological mechanisms for translational research. Designing multi-MPS platforms is essential to study multi-organ systems. Typical design approaches, including direct and allometric scaling, scale each MPS individually and are based on relative sizes not function. This study's aim was to develop a new multi-functional scaling approach for integrated multi-MPS platform design for specific applications. We developed an optimization approach using mechanistic modeling and specification of an objective that considered multiple MPS functions, e.g., drug absorption and metabolism, simultaneously to identify system design parameters. This approach informed the design of two hypothetical multi-MPS platforms consisting of gut and liver (multi-MPS platform I) and gut, liver and kidney (multi-MPS platform II) to recapitulate in vivo drug exposures in vitro. This allows establishment of clinically relevant drug exposure-response relationships, a prerequisite for efficacy and toxicology assessment. Design parameters resulting from multi-functional scaling were compared to designs based on direct and allometric scaling. Human plasma time-concentration profiles of eight drugs were used to inform the designs, and profiles of an additional five drugs were calculated to test the designed platforms on an independent set. Multi-functional scaling yielded exposure times in good agreement with in vivo data, while direct and allometric scaling approaches resulted in short exposure durations. Multi-functional scaling allows appropriate scaling from in vivo to in vitro of multi-MPS platforms, and in the cases studied provides designs that better mimic in vivo exposures than standard MPS scaling methods., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.). Microphysiological Systems Program (4-UH3-TR000496-03)

Epithelial organoids are now an important tool in fields ranging from regenerative medicine to drug discovery. Organoid culture requires Matrigel, a complex, tumor-derived, extracellular matrix. An alternative completely synthetic matrix could improve culture reproducibility, clarify mechanistic phenomena, and enable applications involving human implantation. Here, we designed synthetic matrices with tunable biomolecular and biophysical properties that allowed us to identify critical gel parameters in organoid formation. Inspired by known epithelial integrin expression in the proliferative niche of the human intestine, we identified an α2β1 integrin-binding peptide as a critical component of the synthetic matrix that supports human duodenal colon and endometrial organoid propagation. We show that organoids emerge from single cells, retain their proliferative capacity, are functionally responsive to basolateral stimulation and have correct apicobasal polarity upon induction of differentiation. The local biophysical presentation of the cues, rather than bulk mechanical properties, appears to be the dominant parameter governing epithelial cell proliferation and organoid formation in the synthetic matrix.

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 μL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples., National Institutes of Health (Grant R01 EB010246)

The most clinically relevant risk factor for Clostridioides difficile-associated disease (CDAD) is recent antibiotic treatment. Though most broad-spectrum antibiotics significantly disrupt the structure of the gut microbiota, only particular ones increase CDAD risk, suggesting additional factors might increase the risk from certain antibiotics. Here we show that commensal-independent effects of antibiotics collectively prime an in vitro germ-free human gut for CDAD. We found a marked loss of mucosal barrier and immune function with CDAD-associated antibiotic pretreatment distinct from pretreatment with an antibiotic unassociated with CDAD, which did not reduce innate immune or mucosal barrier functions. Importantly, pretreatment with CDAD-associated antibiotics sensitized mucosal barriers to C. difficile toxin activity in primary cell-derived enteroid monolayers. These data implicate commensal-independent host changes in the increased risk of CDAD with specific antibiotics. Our findings are contrary to the previously held belief that antibiotics allow for CDAD solely through disruption of the microbiome. We anticipate this work to suggest potential avenues of research for host-directed treatment and preventive therapies for CDAD, and to impact human tissue culturing protocols.

An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the N-linked protein glycosylation pathway in Campylobacter jejuni pathogenicity. The gut-immune co-culture (GIC) was developed to model important aspects of the human small intestine by the inclusion of mucin-producing goblet cells, human enterocytes and dendritic cells, bringing together a mucus-containing epithelial monolayer with elements of the innate immune system. The utility of the system was demonstrated by characterizing host–pathogen interactions facilitated by N-linked glycosylation, such as host epithelial barrier functions, bacterial invasion and immunogenicity. Changes in human intestinal barrier functions in the presence of 11168 C. jejuni (wildtype) strains were quantified using GICs. The glycosylation-impaired strain 11168 ΔpglE was 100-fold less capable of adhering to and invading this intestinal model in cell infectivity assays. Quantification of inflammatory signaling revealed that 11168ΔpglE differentially modulated inflammatory responses in different intestinal microenvironments, suppressive in some but activating in others. Virulence-associated outer membrane vesicles produced by wildtype and 11168ΔpglE C. jejuni were shown to have differential composition and function, with both leading to immune system activation when provided to the gut-immune co-culture model. This analysis of aspects of C. jejuni infectivity in the presence and absence of its N-linked glycome is enabled by application of the gut-immune model, and we anticipate that this system will be applicable to further studies of C. jejuni and other enteropathogens of interest.

Bacteroides thetaiotaomicron is a human-associated bacterium that holds promise for delivery of therapies in the gut microbiome1. Therapeutic bacteria would benefit from the ability to turn on different programs of gene expression in response to conditions inside and outside of the gut; however, the availability of regulatory parts, and methods to combine them, have been limited in B. thetaiotaomicron2-5. We report implementation of Cello circuit design automation software6 for this species. First, we characterize a set of genome-integrated NOT/NOR gates based on single guide RNAs (CRISPR-dCas9) to inform a Bt user constraint file (UCF) for Cello. Then, logic circuits are designed to integrate sensors that respond to bile acid and anhydrotetracycline (aTc), including one created to distinguish between environments associated with bioproduction, the human gut, and after release. This circuit was found to be stable under laboratory conditions for at least 12 days and to function in bacteria associated with a primary colonic epithelial monolayer in an in vitro human gut model system.

Three-dimensional micro-physiological in vitro representations of human tissues and organs are emerging as important models of human pathophysiology and stand to make a significant impact on the process of drug discovery and development. An enduring need is to create microvascular networks within such 3D models, particularly for tissues with high metabolic demand such as the liver, pancreas, and the central nervous system. Here we report a facile approach to drive angiogenesis in nascent 3D culture models by embedding degradable hydrogel microbeads coated with induced pluripotent stem cell-derived endothelial cells (MB-iPSC-ECs) in a dense epithelial tissue. Specifically, we describe an approach to optimize microbead scaffold cues, independent of the external environment, by evaluating the iPSC-EC to microbead adhesion properties and how they influence the propensity of cells to both coat microbeads uniformly and undergo sprouting angiogenesis. We encapsulated MB-iPSC-ECs in PEG hydrogels, systematically varied the relative concentration of integrin-targeting peptide motifs in the microbead and surrounding environment, and found that an optimal microbead scaffold ligand regime of 0.1–0.25 mM promotes iPSC-EC monolayer formation and subsequent invasion into the synthetic matrix. We used these results to predict the regime of adhesion ligand required to promote angiogenesis of MB-iPSC-ECs in a co-culture hepatocarcinoma (HEPG2) microtissue model. This modular degradable microbead platform has the potential to promote angiogenic sprouting, which may ultimately support vascularization of a variety of cell-dense tissues.

In this work, we first describe the population variability in hepatic drug metabolism using cryopreserved hepatocytes from five different donors cultured in a perfused three-dimensional human liver microphysiological system, and then show how the resulting data can be integrated with a modeling and simulation framework to accomplish in vitro-in vivo translation. For each donor, metabolic depletion profiles of six compounds (phenacetin, diclofenac, lidocaine, ibuprofen, propranolol, and prednisolone) were measured, along with metabolite formation, mRNA levels of 90 metabolism-related genes, and markers of functional viability [lactate dehydrogenase (LDH) release, albumin, and urea production]. Drug depletion data were analyzed with mixed-effects modeling. Substantial interdonor variability was observed with respect to gene expression levels, drug metabolism, and other measured hepatocyte functions. Specifically, interdonor variability in intrinsic metabolic clearance ranged from 24.1% for phenacetin to 66.8% for propranolol (expressed as coefficient of variation). Albumin, urea, LDH, and cytochrome P450 mRNA levels were identified as significant predictors of in vitro metabolic clearance. Predicted clearance values from the liver microphysiological system were correlated with the observed in vivo values. A population physiologically based pharmacokinetic model was developed for lidocaine to illustrate the translation of the in vitro output to the observed pharmacokinetic variability in vivo. Stochastic simulations with this model successfully predicted the observed clinical concentration-time profiles and the associated population variability. This is the first study of population variability in drug metabolism in the context of a microphysiological system and has important implications for the use of these systems during the drug development process., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (Grant W911NF-12-2-0039), National Institutes of Health (U.S.). Microphysiological Systems Program (Grant 4-UH3-TR000496-03)

The liver is a highly metastasis-permissive organ, tumor seeding of which usually portends mortality. Its unique and diverse architectural and cellular composition enable the liver to undertake numerous specialized functions, however, this distinctive biology, notably its hemodynamic features and unique microenvironment, renders the liver intrinsically hospitable to disseminated tumor cells. The particular focus for this perspective is the bidirectional interactions between the disseminated tumor cells and the unique resident cell populations of the liver; notably, parenchymal hepatocytes and non-parenchymal liver sinusoidal endothelial, Kupffer, and hepatic stellate cells. Understanding the early steps in the metastatic seeding, including the decision to undergo dormancy versus outgrowth, has been difficult to study in 2D culture systems and animals due to numerous limitations. In response, tissue-engineered biomimetic systems have emerged. At the cutting-edge of these developments are ex vivo ‘microphysiological systems’ (MPS) which are cellular constructs designed to faithfully recapitulate the structure and function of a human organ or organ regions on a milli- to micro-scale level and can be made all human to maintain species-specific interactions. Hepatic MPSs are particularly attractive for studying metastases as in addition to the liver being a main site of metastatic seeding, it is also the principal site of drug metabolism and therapy-limiting toxicities. Thus, using these hepatic MPSs will enable not only an enhanced understanding of the fundamental aspects of metastasis but also allow for therapeutic agents to be fully studied for efficacy while also monitoring pharmacologic aspects and predicting toxicities. The review discusses some of the hepatic MPS models currently available and although only one MPS has been validated to relevantly modeling metastasis, it is anticipated that the adaptation of the other hepatic models to include tumors will not be long in coming.

Stem cell use for tissue replacement is limited, owing to rapid death induced in the hostile wound environment. This study found that restricting epidermal growth factor (EGF) receptor signaling to the membrane provided a survival advantage. A method is proposed to tether EGF to bone induction material to improve the survival of mesenchymal stem cells/multipotent stromal cells in vivo., Mesenchymal stem cells/multipotent stromal cells (MSCs) are attractive candidates for cell therapies owing to their ability to differentiate into many lineages. However, these cells often fail to survive when implanted into a harsh wound environment, limiting efficacy in vivo. To improve MSC survival, we previously found that tethered epidermal growth factor (tEGF) molecules that restrict epidermal growth factor receptor (EGFR) signaling to the cell surface provide resistance to death signals. To adapt this system to wound healing, we tethered epidermal growth factor (EGF) to tricalcium phosphate (TCP) particle scaffolds, clinically used in bone healing. Human primary MSCs seeded on TCP and mixed into a collagen-based gel were injected in the perifascial space of immunocompetent mice with or without tEGF attached to the surface. We found that tethering EGF to the TCP scaffolds yielded approximately a fourfold increase in MSC survival compared with non-EGF scaffolds at 21 days, as well as significant improvements in survival in the short term at 2 and 7 days after implantation. Overall, our approach to sustaining EGFR signaling reduced MSC death in vivo and may be useful for future cell therapies where MSCs typically die on implantation. Significance Stem cells are limited as tissue replacements owing to rapid death induced in the hostile wound environment. It has been found that restricting epidermal growth factor (EGF) receptor signaling to the membrane provides a survival advantage. This report elucidates a method to tether EGF to bone induction material to improve the survival of mesenchymal stem cells/multipotent stromal cells in vivo.